Evaluation of LC-MS and LC×LC-MS in analysis of zebrafish embryo samples for comprehensive lipid profiling

Abstract

In this study, both conventional one-dimensional liquid chromatography (1DLC) and comprehensive two-dimensional liquid chromatography (2DLC) coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF MS) were used for full-scale lipid characterization of lipid extracts from zebrafish embryos. We investigated the influence on annotated lipids and different separation mechanisms (HILIC, C18, and PFP), and their different orders arranged in the first and the second dimensions. As a result, the number of lipid species annotated by conventional one-dimensional LC-MS was between 212 and 448. In contrast, the number of individual lipids species annotated by C18×HILIC, HILIC×C18, and HILIC×PFP were 1784, 1059, and 1123, respectively. Therefore, it was evident that the performance of comprehensive 2DLC, especially the C18×HILIC method, considerably exceeded 1DLC. Interestingly, a comparison of the HILIC×C18 and C18×HILIC approaches showed, under the optimized conditions, similar orthogonality, but the effective separation power of the C18×HILIC was much higher. A comparison of the HILIC×C18 and the HILIC×PFP methods demonstrated that the HILIC×PFP separation had superior orthogonality with a small increase on its effective peak capacity, indicating that the HILIC×PFP combination maybe a promising platform for untargeted lipidomics in complex samples. Finally, from the comprehensive lipid profiling respective, the C18×HILIC was selected for further studies.

Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts

“…Single-phase extraction by adding more of the same solvent used for protein precipitation (e.g., methanol (MeOH) and/or acetonitrile (ACN) after removing the precipitate) is commonly applied [22,69]. However, to extract and dissolve non-polar lipids, a more apolar organic solvent, such as methyl tert-butyl ether (MTBE), dichloromethane (CH 2 Cl 2 ), or chloroform (CHCl 3 ), is usually required [34,70]. Single-phase methods are attractive because they reduce the time and complexity of the extraction, but they are also subject to a higher matrix effect and a smaller detection range due to the polarity diversity of the molecules in the metabolome (e.g., the LogP for citric acid is −1.64, while the predicted LogP (XLogP) for lysophosphatidylcholine (18:0) and triacylglycerol (48:0) are 6.6 and 22.1, respectively) [10,40,71,72].…”

“…Keerthisinghe et al used a HILIC Luna aminopropyl column (150 × 10 mm, 3 µm) for polar metabolites in positive and negative ionization modes and an RPLC (C18) Zorbax Eclipse Plus RRHD column (50 × 2.1 mm, 1.8 µm) in positive mode for lipidomics [48]. Recently, Xu et al analyzed zebrafish embryos using different combinations of chromatographic methods to evaluate their metabolite coverage [70,102]. Their results highlighted the need for two methods, one HILIC based with an XBridge Amide column (150 × 2.1 mm × 3.5 µm) in ESI+ and one pentafluorophenyl Kinetex F5 column (150 × 2.1 mm × 2.6 µm) in ESI− to cover the polar metabolome (336 annotated metabolites) and one comprehensive two-dimensional (2D) liquid chromatography method with an EVO C18 column (100 × 2.1 mm × 2.6 µm) and a BEH HILIC column (50 × 2.1 mm × 1.7 µm) for comprehensive lipid profiling of zebrafish embryos (1784 annotated lipids).…”

Recurrent Topics in Mass Spectrometry-Based Metabolomics and Lipidomics—Standardization, Coverage, and Throughput

“…Adoption of comprehensive LC X LC-MS in metabolomics and lipidomics has been limited mainly because comprehensive 2D-LC-MS metabolomics approaches developed up to date have largely suffered from incomplete usage of separation space in HILIC and RP-LC combinations and from severe sensitivity loss. 247 The latter diminishes actual coverage in nontargeted screenings. Solvent evaporation interfaces as featured in SFC X RP-LC lipidomics might overcome this challenge.…”

Mass Spectrometry-Based Zebrafish Toxicometabolomics: A Review of Analytical and Data Quality Challenges

“…Single-phase extraction by adding more of the same solvent used for protein precipitation (e.g., methanol (MeOH) and/or acetonitrile (ACN) after removing the precipitate) is commonly applied [ 22 , 69 ]. However, to extract and dissolve non-polar lipids, a more apolar organic solvent, such as methyl tert-butyl ether (MTBE), dichloromethane (CH₂Cl₂), or chloroform (CHCl 3 ), is usually required [ 34 , 70 ]. Single-phase methods are attractive because they reduce the time and complexity of the extraction, but they are also subject to a higher matrix effect and a smaller detection range due to the polarity diversity of the molecules in the metabolome (e.g., the LogP for citric acid is −1.64, while the predicted LogP (XLogP) for lysophosphatidylcholine (18:0) and triacylglycerol (48:0) are 6.6 and 22.1, respectively) [ 10 , 40 , 71 , 72 ].…”

“…Keerthisinghe et al used a HILIC Luna aminopropyl column (150 × 10 mm, 3 μm) for polar metabolites in positive and negative ionization modes and an RPLC (C18) Zorbax Eclipse Plus RRHD column (50 × 2.1 mm, 1.8 μm) in positive mode for lipidomics [ 48 ]. Recently, Xu et al analyzed zebrafish embryos using different combinations of chromatographic methods to evaluate their metabolite coverage [ 70 , 102 ]. Their results highlighted the need for two methods, one HILIC based with an XBridge Amide column (150 × 2.1 mm × 3.5 μm) in ESI+ and one pentafluorophenyl Kinetex F5 column (150 × 2.1 mm × 2.6 μm) in ESI− to cover the polar metabolome (336 annotated metabolites) and one comprehensive two-dimensional (2D) liquid chromatography method with an EVO C18 column (100 × 2.1 mm × 2.6 μm) and a BEH HILIC column (50 × 2.1 mm × 1.7 μm) for comprehensive lipid profiling of zebrafish embryos (1784 annotated lipids).…”